•During the past years PCR has become the gold standard for the molecular diagnostics of manifold diseases, such as cancer or infectious diseases. In most cases real-time PCR applications have replaced conventional PCR tests due to their obvious benefits, namely speed, increased specificity, (semi-) quantification, reduced work-load, and a minimized risk of carry-over contamination, which is of benefit, especially in diagnostic labs. Troubleshooting of the failed PCR reaction was Human error, Failure of the positive control, Primer and probe damage, problems with the basic reaction mix from the manufacturer, the inhibiting influence of water, individual problems with the cycler, problems with the machine, and the problem with the pathogen. Preparing important PCR assay protocols for reagents from more than one manufacturer may help to ensure rapid reliable diagnostics.